The use of light scattering measurements as a means for differentiating various types of small particles is well known. For example, in virtually all sophisticated hematology instruments, it is common to measure the forward light scattering properties of blood cells by passing the cells, one at a time, through the interrogation zone of an optical flow cell. While in the interrogation zone, each cell is irradiated by a laser beam, and one or more photodetectors, strategically positioned forward of the interrogation zone, operate to sense the level of forward scattered radiation, often within different predetermined angular ranges. In addition to measuring forward light scatter, some hematology instruments measure side scatter as well, using a separate photodetector located laterally of the irradiated cell. These light scattering measurements are often combined with other simultaneously made measurements, e.g., axial light-loss, DC volume and/or RF conductivity measurements, to better differentiate cell types of particular interest from other cells and particulate material in the sample that have similar light-scattering properties within the measurement ranges. Having made the various parameter measurements, the instrument then produces scattergrams in which the different parameters measured are plotted against each other. Ideally, each cell type appears on these scattergrams as a tight cluster of data points, each point representing an individual cell, and each cluster being readily identifiable by a clearly identified spacing from other clusters of data points. In such case, it is a relatively simple matter to “gate” cells of one cluster from those of another cluster and to enumerate the cells of each type. This ideal, unfortunately, is sometimes difficult to realize since, for a variety of reasons, a certain (low) percentage of cells of one type invariably invade the spatial domain of cells of other types, making the differentiation of each type somewhat imprecise.
It has been suggested that multiple bundles of fiber optics, arranged in concentric rings, can be used to optically couple scattered radiation from a scatter plane to multiple photodetectors (e.g., photomultiplier tubes and photodiodes) remotely spaced from the scatter plane. See, “Cell Differentiation Based on Absorption and Scattering” by Wolfgang G. Eisert, The Journal of Histology and Cytochemistry, Vol.27, No.1, pp404-409 (1979). As described by Eisert, optical fibers are arranged so that their respective light-collecting ends form five concentric rings centered about a centrally located light-collecting bundle of optical fibers. The respective distal ends of the individual fibers of each of the five concentric rings are optically coupled to five different photomultiplier tubes, and the distal ends of the individual fibers of the center bundle are optically coupled to a photodiode. Thus, each ring of fibers collects scattered light in a discrete angular range determined by the diameter of the fiber (or the width of the rings), the radial displacement of the fiber end relative to the beam axis (i.e., the diameter of the ring), and the axial spacing of the fiber ends from the scattering light source. The center bundle of fibers is optically aligned with the beam axis, and the other bundles, with their individual fibers being arranged in a circle, are arranged parallel to the beam axis. The center bundle of fiber optics, being positioned on the beam axis, serves to monitor the axial light loss of the beam, as occasioned by the passage of cells therethrough.
In the fiber-optic light coupler proposed by Eisert above, the respective light-collecting ends of all the fibers are disposed in a common plane that is arranged perpendicular to the optical axis of the cell-irradiating light beam. Thus, it will be appreciated that, due to the numerical aperture of the fibers, the optical coupling of scattered light into the optical fibers deteriorates as the scatter angle increases. Additionally, as the scatter angle increases, the angle of incidence between the scattered light and the fiber end increases, thereby increasing the number of internal reflections required to transmit the scattered light from one end of the fiber to the other end. This problem of coupling efficiency is exacerbated by the dramatic reduction in scatter intensity at relatively large scatter angles.
In addition to forward- and side-scatter measurements, it has been suggested that back-scatter (i.e., reflected light) measurements may prove useful in differentiating blood cell types. In a theoretical paper entitled, “Elastic Light Scattering From Nucleated Blood Cells: Rapid Numerical Analysis,” by Sloot and Figdor, Applied Optics, Vol. 25, No. 19, 1 Oct. 1986, it is noted that simultaneous detection of the light-scatter intensities in the forward-, lateral-, and backscatter directions is required to optimize the detection of different cell types in heterogeneous populations of nucleated blood cells. Here, a model is presented to calculate the light-scattering properties of nucleated blood cells which are mimicked by two concentric spheres. It is derived from the calculations presented (no actual measurements on cells were made) that the back-scatter intensity is determined by the nucleus/cytoplasm ratio and changes in the optical density of the cytoplasm and nucleus. The analysis presented strongly suggests a direct correlation between the transparency of the nucleus and the intensity of the back-scatter signal. While no hardware is disclosed in this paper for making any light-scattering measurements at all, a subsequent paper, “Scattering Matrix Elements of Biological Particles Measured in a Flow Through System: Theory and Practice” by Sloot et al., Applied Optics, Vol. 28, No. 10, 15 May 1989, alludes to the use of large surface-area scatter detectors and the need to apply “large cone integration” to account for the relatively large surfaces. This paper schematically illustrates a back-scatter detector having a central aperture through which a particle-irradiating laser beam travels before irradiating the particle. Upon striking the particle, the large surface of the back-scatter detector collects and detects back-scattered light through a large cone angle, i.e., throughout a large angular range. While large surface area detectors are advantageous from the standpoint that they produce a relatively strong signal due to their collection of light scattered over a large angular range, they are disadvantageous from a signal-to-noise standpoint. As the surface area increases, the detector is sensitive to increasing amounts of stray light, e.g. the laser light reflecting from the faces of the optical flow cell through which the back-scattered light from the cells is detected.